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C Bruce A Whitelaw

Publications and source records attributed to C Bruce A Whitelaw.

11 recordsLinked to original sources
PubMed2006

In Vitro and in Vivo nucleosome positioning on the ovine beta-lactoglobulin gene are related.

Although positioned nucleosomes are known to play a direct, localised role in regulating access to DNA sequence, they also have the potential, through their long-range distribution, to affect the detailed structure of the higher-order chromatin fibre. To investigate this possibility, we firstly mapped, in vitro, the sequence-dependent positions that the core histone octamer adopts when reconstituted onto DNA containing the ovine beta-lactoglobulin gene. These positioning sites are discussed in terms of their relative affinity for the histone octamer, their locations with respect to the gene sequence and their periodic distribution throughout the gene region. Secondly, we mapped, in vivo, the sites that nucleosomes occupy on the same sequence in liver nuclei, where the gene is transcriptionally inactive. Although the sequence is largely packaged into regularly spaced nucleosomes, reflecting a fibre of uniform higher-order structure, this organisation is disrupted by a number of unusual chromatin structures in a region stretching from the second to the third introns of the gene. A comparison of the in vitro and in vivo nucleosome positioning data shows that they are qualitatively and quantitatively related, suggesting that the structure of the higher-order chromatin fibre containing the beta-lactoglobulin gene is determined, in part, by the long-range organisation of the non-coding sequences within which the gene is embedded.

Animals↗
PubMed2006

A virus-neutralising antibody is not cytotoxic in vitro.

Hybridoma cell lines are characterized by a preferential loss of the heavy chain gene. This observation has led to the theory that the immunoglobulin heavy chain possesses an intrinsic cytotoxic activity in some cell types. We have generated transgenic mice expressing the heavy and light chain genes of the virus-neutralising antibody A1 carrying constant domains of the human gamma1 and kappa isotype. Heavy chain and light chain transgenes were under trancriptional control of identical promoter regions derived from the mammary gland specific ovine beta-lactoglobulin gene. The copy number of the heavy chain transgene was consistently lower than the copy number of the light chain gene in all lines of transgenic mice. Moreover, the light chain gene was expressed in significant excess of the heavy chain gene in the lactating mammary gland in all transgenic lines. In several transgenic lines, the differences in antibody expression were greater than could be explained by the differences in transgene copy number. One potential cause of this phenotype could be a cytotoxic effect of free heavy chain protein in embryonic cells (resulting in differences in copy number) or mammary epithelial cells (resulting in differences in transgene expression). We therefore directly assessed the effect of the expression of free A1 heavy chain protein in epithelial cell lines and in murine embryonic stem cells. However, full-length A1 heavy chain mRNA and protein could be expressed transiently and stably in both epithelial and embryonic stem cells and had no detectable effect on cell viability. Taken together, these findings argue against an inherent cytotoxicity of the free A1 heavy chain protein in epithelial or embryonic cells.

Animals↗
PubMed2005

Duplication of Stat5-binding sites within the beta-lactoglobulin promoter compromises transcription in vivo.

Achieving high and reliable levels of expression is essential for biotechnological application of transgene technology. In an attempt to maximise transgene expression, we analysed the effect of adding extra Stat5-binding sites, while maintaining their spatial arrangement, to the beta-lactoglobulin promoter. A 195 bp-fragment comprising the three Stat5-binding sites in the beta-lactoglobulin promoter was duplicated directly upstream, 88 bp or 177 bp upstream from the original site. We assessed the effect that addition of extra Stat5-binding sites had in transcriptional activation and chromatin structuring of the promoter, both in vitro in HC11 cells and in vivo in transgenic animals. Duplication of Stat5-binding sites in the beta-lactoglobulin promoter did favour chromatin remodelling in vivo but unexpectedly did not enhance transcriptional activation of the transgene, despite the higher basal and induced expression levels achieved in vitro in HC11 cells. Furthermore, the well-described positive relationship between beta-lactoglobulin transgene copy number and mammary expression was lost in the new lines. These results indicate that chromatin remodelling upon Stat5 binding is not sufficient to activate transgene expression, denote that strategies to achieve transgenic transcriptional activation are not obvious.

Animals↗
PubMed2005

Live lambs born from zona-pellucida denuded embryos.

Progress with techniques using zona-pellucida denuded embryos has resulted in the birth of live cattle, pigs, and mice. The application of zona-free methods in sheep has been restricted to in vitro studies. In this report, we demonstrate that live lambs can be produced from zona-free IVF embryos. We are pursuing this method as a prerequisite to developing viral vector co-culture delivery strategies.

Animals↗
PubMed2004

Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector.

Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV-1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.

Animals↗
PubMed2003

Mono-allelic expression of variegating transgene locus in the mouse.

We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation.

Alleles↗
PubMed2002

Multiple effects of genetic background on variegated transgene expression in mice.

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.

Animals↗
PubMed2002

Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice.

We have previously demonstrated that IGFBP-5 production by mammary epithelial cells increases dramatically during involution of the mammary gland. To demonstrate a causal relationship between IGFBP-5 and cell death we created transgenic mice expressing IGFBP-5 in the mammary gland using a mammary-specific promoter, beta-lactoglobulin. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Histological analysis indicated reduced numbers of alveolar end buds, with decreased ductal branching. Transgenic dams produced IGFBP-5 in their milk at concentrations similar to those achieved at the end of normal lactation. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. BrdU labelling was decreased, whereas DNA ladders were increased in transgenic animals on day 1 of lactation. On day 2 postpartum, the epithelial invasion of the mammary fat pad was clearly impaired in transgenic animals. The concentrations of the pro-apoptotic molecule caspase-3 and of plasmin were both increased in transgenic animals whilst the concentrations of 2 prosurvival molecules Bcl-2 and Bcl-x(L)were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I we examined IGF receptor phosphorylation and Akt phosphorylation and showed that both were inhibited. We attempted to "rescue" the transgenic phenotype by using growth hormone to increase endogenous IGF-I concentrations or by implanting minipumps delivering an IGF-1 analogue, R(3)-IGF-1, which binds weakly to IGFBP-5. Growth hormone treatment failed to affect mammary development suggesting that increased concentrations of endogenous IGF-1 are insufficient to overcome the high concentrations of IGFBP-5 produced by these transgenic animals. In contrast mammary development (gland weight and DNA content) was normalised by R3-IGF-I although milk production was only partially restored. This is the first demonstration that over-expression of IGFBP-5 can lead to; impaired mammary development, increased expression of the pro-apoptotic molecule caspase-3, increased plasmin generation and decreased expression of pro-survival molecules of the Bcl-2 family. It clearly demonstrates that IGF-I is an important developmental/survival factor for the mammary gland and, furthermore, this cell death programme may be utilised in a wide variety of tissues.

Animals↗
PubMedDate not supplied

Comparative analysis on the structural features of the 5' flanking region of kappa-casein genes from six different species.

Kappa-casein plays an essential role in the formation, stabilisation and aggregation of milk micelles. Control of kappa-casein expression reflects this essential role, although an understanding of the mechanisms involved lags behind that of the other milk protein genes. We determined the 5'-flanking sequences for the murine, rabbit and human kappa-casein genes and compared them to the published ruminant sequences. The most conserved region was not the proximal promoter region but an approximately 400 bp long region centred 800 bp upstream of the TATA box. This region contained two highly conserved MGF/STAT5 sites with common spacing relative to each other. In this region, six conserved short stretches of similarity were also found which did not correspond to known transcription factor consensus sites. On the contrary to ruminant and human 5' regulatory sequences, the rabbit and murine 5'-flanking regions did not harbour any kind of repetitive elements. We generated a phylogenetic tree of the six species based on multiple alignment of the kappa-casein sequences. This study identified conserved candidate transcriptional regulatory elements within the kappa-casein gene promoter.

5' Flanking Region↗